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Csk inhibition of c-Src activity requires both the SH2 and SH3 domains of Src
G Superti-Furga1, S Fumagalli, M Koegl
1European Molecular Biology Laboratory, Heidelberg, Germany.
Abstract:
The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its carboxy-terminal tail. A kinase that phosphorylates Tyr527, called Csk, has recently been identified. We expressed c-Src in yeast to test the role of the SH2 and SH3 domains of Src in the negative regulation exerted by Tyr527 phosphorylation. Inducible expression of c-Src in Schizosaccharomyces pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in either the SH2 or SH3 domain were as lethal as wild type c-Src, but were insensitive to Csk, even though they were substrates for Csk in vivo. Peptide binding experiments revealed that Src proteins with mutant SH3 domains adopted a conformation in which the SH2 domain was not interacting with the tail. These data support the model of an SH2 domain-phosphorylated tail interaction repressing c-Src activity, but expand it to include a role for the SH3 domain. We propose that the SH3 domain contributes to the maintenance of the folded, inactive configuration of the Src molecule by stabilizing the SH2 domain-phosphorylated tail interaction. Moreover, the system we describe here allows for further study of the regulation of tyrosine kinases in a neutral background and in an organism amenable to genetic analysis.
Insights
The protein tyrosine kinase c-Src is regulated by its tail phosphorylation. Yeast studies reveal the SH3 domain stabilizes this interaction, maintaining c-Src inactivity and offering a new model for kinase regulation.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Protein tyrosine kinase c-Src activity is regulated by phosphorylation at Tyr527.
- The kinase Csk phosphorylates Tyr527, leading to negative regulation of c-Src.
- The roles of Src's SH2 and SH3 domains in this regulatory mechanism require further elucidation.
Purpose of the Study:
- To investigate the function of SH2 and SH3 domains in c-Src negative regulation by Tyr527 phosphorylation.
- To establish a yeast-based system for studying tyrosine kinase regulation.
Main Methods:
- Expression of wild-type and mutant c-Src in Schizosaccharomyces pombe.
- Co-expression of Csk to assess rescue of c-Src-induced lethality.
- Peptide binding assays to analyze domain interactions.
Main Results:
- Inducible c-Src expression in yeast caused cell death, which was counteracted by Csk co-expression.
- Mutations in SH2 or SH3 domains rendered c-Src insensitive to Csk, despite being phosphorylated in vivo.
- Mutant SH3 domains altered conformation, disrupting SH2 domain interaction with the tail.
Conclusions:
- The SH2 domain-phosphorylated tail interaction is crucial for c-Src repression.
- The SH3 domain plays a role in maintaining the inactive conformation by stabilizing the SH2-tail interaction.
- A yeast system provides a valuable platform for studying tyrosine kinase regulation.